Methods, Systems and Apparatus for Separating Components of a Biological Sample

ABSTRACT

Described herein are methods, systems and apparatus for separating components of a biological sample; as well as methods of using compositions prepared by same. In some embodiments, the present invention provides a method for separating components of a biological sample, the method comprising: introducing a biological sample having a plurality of components to a tube comprising: a lumen; a proximal end; a distal end; an interior wall; and an exterior wall; applying a force to said tube for a time sufficient to separate said plurality of components; and agitating said tube at an angle of from about 5° to about 60°.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority from U.S. ProvisionalApplication No. 62/794,961, filed Jan. 21, 2019; the contents of whichare hereby incorporated herein in their entirety.

BACKGROUND

Platelet-rich plasma (PRP) is generally understood to be a concentrateof platelets and plasma, that also contains growth factors, such asPlatelet-Derived Growth Factor (PDGF); Transforming Growth Factor group(TGF); Epidermal Growth Factor (EGF); Vascular Endothelial Growth Factor(VEGF); Fibroblast Growth Factor (FGF); and Keratinocyte Growth Factor(KGF), which regulate the healing cascade by signaling surrounding cellsto repair damaged tissue and regenerate new tissue.

Various methods and systems for preparing PRP are known; but for avariety of reasons, these methods and systems do not consistentlyprovide efficient platelet capture. For example, devices and systemsutilizing a separator gel, tend to have issues with platelets adheringto the separator gel. As a result, the clinician is often left with aless than desirable number of platelets available for administration toa patient.

Thus, there remains a need for simple, cost-effective, reliable andclinically useful methods for overcoming the aforementioned challenges;and that enrich platelet concentrations and increase the number ofplatelets available for administration to a patient. Embodiments of thepresent invention are designed to meet these and other ends.

SUMMARY

In some embodiments, the claimed invention is directed to methods forseparating components of a biological sample, the method comprising:introducing a biological sample having a plurality of components to atube comprising: a lumen; a proximal end; a distal end; an interiorwall; and an exterior wall; applying a force to said tube for a timesufficient to separate said plurality of components; and agitating saidtube at an angle (e.g. from about 5° to about 60°) effective to enrichthe concentration of a component of the biological sample (e.g.platelets).

Other embodiments provide compositions comprising a product produced byany one of the methods or systems described herein. While otherembodiments provide methods of using a product produced by any one ofthe methods or systems described herein

Still further embodiments provide system for separating components of abiological sample comprising: a biological sample; a tube; a means forapplying a centrifugal force to said tube; and a means for agitatingsaid tube.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an exemplary tube of the present invention aftercentrifugation.

FIG. 2 depicts a first comparative agitation angle.

FIG. 3 depicts a second comparative agitation angle.

FIG. 4 depicts a first exemplary agitation angle according to certainembodiments of the present invention.

FIG. 5 depicts a second exemplary agitation angle according to certainembodiments of the present invention.

FIG. 6 depicts a third exemplary agitation angle according to certainembodiments of the present invention.

FIG. 7 illustrates the foam created by an exemplary embodiment of thepresent invention.

FIG. 8 illustrates the foam created by another exemplary embodiment ofthe present invention.

FIG. 9 illustrates the absence of foam when a comparative method isperformed.

DETAILED DESCRIPTION

In some embodiments, the present invention provides a method forseparating components of a biological sample, the method comprising:introducing a biological sample having a plurality of components to atube comprising: a lumen; a proximal end; a distal end; an interiorwall; and an exterior wall; applying a force to said tube for a timesufficient to separate said plurality of components; and agitating saidtube at an angle of from about 5° to about 60°. In some embodiments, theforce is a centrifugal force.

As part of the Instructions for Use (IFU), PRP systems typically callfor a gentle inversion of the collection tube following centrifugation.The inversion allows for resuspension of the platelets in the sample ofPRP.

In some embodiments, the present invention provides a method wherein atube containing a separated biological sample is agitated along itslongitudinal axis in a rapid manner at a rate of several times persecond. In some embodiments, the method may be performed for a fewseconds up to one minute. In certain embodiments, the agitation anglemay be slightly negative (−15 degrees) to vertical (+90 degrees).

Without being bound by theory, the present inventors believe that themethods of the present invention create a washing (i.e., lavage of thesurface of the separation barrier) that helps to release platelets thatmay be attached to, or adhere to the surface of the separation barrierthereby increasing the number of platelets available for resuspensionand administration to a subject.

Some embodiments of the present invention provide a tube comprising amaterial selected from: glass; modified poly amide (MPA); polyethyleneterephthalate (PET) and any other material which is inert to abiological sample. In some embodiments, the tube comprises a laminatestructure wherein an exterior wall of the tube is made of a materialdifferent than the interior wall.

In some embodiments, the tube further comprises a stopper. In someembodiments, the stopper comprises a material inert to biologicalsamples. In other embodiments, the stopper comprises a material thatdoes not crumble. In certain embodiments, the stopper comprises butylrubber or its halo derivative formulations. In further embodiments, thestopper has a hardness of from about forty (40) to sixty (60) Shore A.In other embodiments, the stopper has a hardness designed to providestable vacuum for from about eighteen (18) to about twenty-four (24)months.

In some embodiments, the tube is capable of receiving biological samplesof from about four (4) ml to about one hundred (100) ml. In otherembodiments, the tube is designed to receive biological samples of fromabout eight (8) ml to about fifty (50) ml. Still further embodimentsprovide tubes designed to receive biological samples of from about ten(10) ml to about thirty (30) ml. Other embodiments provide tubesdesigned to receive biological samples of from about eleven (11) ml orabout twenty-two (22) ml.

In some embodiments, the tube is selected from: a vacuum, tube, anon-vacuum tube, a plastic tube, a glass tube, a rigid tube, a non-rigidtube, a semi rigid tube and any combination thereof. In someembodiments, the terms “tube”, “collection tube”, “test tube”, and thelike, may be used interchangeably.

In some embodiments, the tube further comprises a gel. In someembodiments, the gel comprises a thixotropic gel. In furtherembodiments, the gel comprises a polymer. In certain embodiments, thegel can be a homopolymer or a co-polymer comprising a combination ofmonomers. In some embodiments, the gel comprises a polyacrylate,polyolefin or polyester.

Still further embodiments provide a gel having a density at 25° C. offrom about 1.03 g/cm³ to about 1.09 g/cm³. While other embodimentsprovide a gel having a density at 25° C. of from about 1.04 g/cm³ toabout 1.07 g/cm³. In some embodiments, the gel has a density at 25° C.of from about 1.05 g/cm³.

In some embodiments, the gel has a viscosity at 30° C. of from about1,000 to about 5,000 cps. In other embodiments, the gel has a viscosityat 30° C. of from about 1,000 to about 4,500 cps. In furtherembodiments, the gel has a viscosity at 30° C. of from about 1,000 toabout 4,000 cps. While other embodiments utilize a gel having aviscosity at 30° C. of from about 1,000 to about 3,500 cps. Stillfurther embodiments provide a gel having a viscosity at 30° C. of fromabout 1,000 to about 3,000 cps. In other embodiments, the gel has aviscosity at 30° C. of from about 1,500 to about 5,00 cps. In furtherembodiments, the gel has a viscosity at 30° C. of from about 2,000 toabout 5,000 cps. While other embodiments utilize a gel having aviscosity at 30° C. of from about 2,500 to about 5,000 cps. Stillfurther embodiments provide a gel having a viscosity at 30° C. of fromabout 3,000 to about 5,000 cps.

Yet other embodiments provide a separation barrier that does notcomprise a gel, e.g. a solid float. In some embodiments, the float cantake on a variety of shapes and may be constructed from a variety ofmaterials. In certain embodiments, the float is comprised of anon-porous material and has a substantially smooth surface. In someembodiments, the separation barrier is selected from a gel; a solidfloat; and a combination thereof.

In some embodiments, the biological sample is autologous. In someembodiments, the biological sample comprises mammalian blood. In someembodiments, the mammalian blood comprises human blood. In someembodiments, the biological sample comprises whole blood.

Still further embodiments provide a biological sample comprising a firstcomponent comprising a plasma fraction and a second component comprisinglymphocytes, monocytes and erythrocytes. In some embodiments, acentrifugal force is applied for a time sufficient to form a barrierbetween the first component and the second component. In otherembodiments, a centrifugal force is applied for a time sufficient toform a barrier between the plasma fraction and the second componentcomprising lymphocytes, monocytes and erythrocytes.

In certain embodiments, the plasma fraction comprises platelets. In someembodiments, the plasma fraction comprises platelet rich plasma (PRP)and platelet poor plasma. In some embodiments, the plasma fractioncomprises PRP and high-concentrated PRP. In some embodiments, the plasmafraction comprises PRP, high-concentrated PRP and ultra-highconcentrated PRP.

Some embodiments further comprise the step of removing at least aportion of the first component. In some embodiments, from abouttwenty-five percent (25%) to about seventy-five percent (90%) of thefirst component is removed, optionally about thirty percent (30%) toabout seventy percent (85%) of the first component is removed, aboutthirty-five percent (35%) to about sixty-five percent (80%) of the firstcomponent is removed, about forty percent (40%) to about sixty percent(75%) of the first component is removed, about forty-five percent (45%)to about fifty-five percent (70%) of the first component is removed,about forty-five percent (50%) to about fifty-five percent (90%) of thefirst component is removed, about fifty percent (50%), about sixtypercent (60%), about seventy percent (70%), about eighty percent (80%),or about ninety percent (90%), of the first component is removed.

In some embodiments, the tube is agitated for a time sufficient toprovide a plasma fraction having a straw color with a pinkish hue. Inother embodiments, the tube is agitated for a time sufficient to providea plasma fraction having a hue angle, h, in the CIELAB system of from310 to 350 degrees. In further embodiments, the tube is agitated for atime sufficient to provide a plasma fraction having a hue angle, h, inthe CIELAB system of from 310 to 345 degrees. In some embodiments, thetube is agitated for a time sufficient to provide a plasma fractionhaving a hue angle, h, in the CIELAB system of from 310 to 340 degrees.In still further embodiments, the tube is agitated for a time sufficientto provide a plasma fraction having a hue angle, h, in the CIELAB systemof from 310 to 335 degrees. While in other embodiments, the tube isagitated for a time sufficient to provide a plasma fraction having a hueangle, h, in the CIELAB system of from 310 to 330 degrees. Still otherembodiments provide methods wherein the tube is agitated for a timesufficient to provide a plasma fraction having a hue angle, h, in theCIELAB system of from 310 to 325 degrees. Yet other embodiments providemethods wherein the tube is agitated for a time sufficient to provide aplasma fraction having a hue angle, h, in the CIELAB system of from 310to 320 degrees.

In some embodiments, the tube is agitated for a time sufficient tocreate a visually perceivable foam layer. In some embodiments, the foamlayer is created on a surface of the plasma fraction. In someembodiments, the appearance of the foam layer correlates with thesuspension of a clinically significant number of platelets in the plasmafraction. In other embodiments, the appearance of the foam is a signalthat a clinically significant number of platelets are available forextraction and administration to a patient.

In some embodiments, the foam layer has a thickness of from about one(1) millimeter to about five (5) millimeters, optionally from about two(2) millimeters to about five (5) millimeters, or three (3) millimetersto about five (5) millimeters. While in other embodiments, the foamlayer has a density of from about 0.01 g/cm³ to about 0.25 g/cm³,optionally from about about 0.05 g/cm³ to about 0.25 g/cm³, about 0.1g/cm³ to about 0.25 g/cm³, about 0.15 g/cm³ to about 0.25 g/cm³, orabout 0.2 g/cm³ to about 0.25 g/cm³.

In some embodiments, the tube is agitated for from about five (5)seconds to about sixty (60) seconds, optionally from about 5 seconds toabout 50 seconds, about 5 seconds to about 45 seconds, about 5 secondsto about 40 seconds, about 5 seconds to about 35 seconds, about 5seconds to about 30 seconds, about 5 seconds to about 25 seconds, about5 seconds to about 20 seconds, about 5 seconds to about 15 seconds, orabout 5 seconds to about 10 seconds.

In some embodiments, the agitation is stepwise. In some embodiments, thestepwise agitation comprises a plurality of five second intervals ofagitation. In other embodiments, the stepwise agitation furthercomprises a break between five second intervals. In certain embodiments,the break is from about 0.1 seconds to about 5 seconds.

In some embodiments, the agitation is a rhythmic motion. In someembodiments, the agitation creates a longitudinal or transversewave-like motion in the biological sample. In some embodiments, theagitation creates a mixed longitudinal and transverse wave-like motionin the biological sample.

In some embodiments, a centrifugal force of from about 500 g to about5000 g is applied to said tube. In other embodiments, a centrifugalforce of from about 750 g to about 5000 g is applied to said tube. Whilein other embodiments, a centrifugal force of from about 1000 g to about5000 g is applied to said tube. In yet other embodiments, a centrifugalforce of from about 1500 g to about 5000 g is applied to said tube. Insome embodiments, a centrifugal force of from about 2000 g to about 5000g is applied to said tube. In some embodiments, a centrifugal force offrom about 2500 g to about 5000 g is applied to said tube. In someembodiments, a centrifugal force of from about 3000 g to about 5000 g isapplied to said tube. In other embodiments, a centrifugal force of fromabout 3000 g to about 4000 g is applied to said tube. While in otherembodiments, a centrifugal force of from about 1500 g to about 2500 g isapplied to said tube.

In some embodiments, the centrifugal force creates a plasma-gelinterface between a surface of the gel and a surface of the plasmafraction. In some embodiments, the plasma-gel interface comprisesplatelets. In certain embodiments, the platelets in the plasma-gelinterface are releasably bound to a surface of the gel. In someembodiments, the agitation releases platelets from the plasma-gelinterface. In some embodiments, the platelets released from theplasma-gel interface are suspended in the plasma fraction.

In some embodiments, the tube further comprises (or contains) ananticoagulant. In some embodiments, the anticoagulant is selected from:a citrate salt (e.g. buffered sodium citrate); an EDTA salt(potassium-ethylenediaminetetra-acid);citrate-theophylline-adenosine-dipyridamole (CTAD); hirudin,benzylsulfonyl-d-Arg-Pro-4-amidinobenzylamide (BAPA); citric/citratedextrose (ACD); heparin; an iodo acetate salt; an oxalate salt; afluoride salt; and a combination of two or more thereof. Certainembodiments of the present invention do involve the use of a tubecomprising an anticoagulant. In such embodiments, the biological samplemay have been pre-treated with anticoagulant or the biological sampledoes not need to be anticoagulated.

Other embodiments provide compositions comprising a product of any oneof the methods or systems described herein. Still further embodimentsprovide for the use of a composition comprising a product of any one ofthe methods or systems described herein for treating or preventingalopecia, bed sores, wrinkles, pain, tendonitis, arthritis, acne,scarring, crow's feet, ligament sprains and tears, and/or skin lesions.

Still further embodiments provide systems for separating components of abiological sample comprising: a biological sample; a tube; a means forapplying a centrifugal force to said tube (e.g. a centrifuge); and ameans for agitating said tube. In some embodiments, the systemsdescribed herein further comprise a means for measuring color in abiological sample. In some embodiments, the means for measuring color ina biological sample is selected from a spectrophotometer and adensitometer.

In some embodiments, the centrifuge is selected from a fixed anglecentrifuge and horizontal spin centrifuge, or a swinging bucketcentrifuge.

In some embodiments, the means for agitating the tube is adapted tolinearly agitate the tube. In some embodiments, the means for agitatingthe tube is a tube rocker.

Some embodiments of the present invention provide a system as describedherein further comprising a platelet counter. While other embodimentsfurther comprise a processor. In some embodiments, the processor iswirelessly coupled to the means for applying a centrifugal force; themeans for agitating the tube; the means for measuring color in abiological sample; and the platelet counter. In some embodiments, themeans for applying a centrifugal force; the means for agitating thetube; the means for measuring color in a biological sample; the plateletcounter; and the processor are contained in a single apparatus.

As used herein, the term “available platelet count” (or “APC”) isintended to refer to the number of platelets that are readily accessibleto the clinician for administration to a subject in need thereof.

In some embodiments, the methods and systems described herein increasethe available platelet count (“APC”) by at least about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, about 100%, about 105%, about 110%,about 115%, about 120%, about 125%, about 130%, about 135%, about 140%,about 145%, about 150%, about 200%, about 250%, about 300%, about 400%,or about 500%, versus the platelet count provided by a control system.In some embodiments, the control system substantially similar systemthose encompassed by the present invention, except for the absence of ameans for agitating the tube; and/or a substantially similar systemwherein the means for agitating the tube is only able to agitate thetube at an angle less than 5°, or at an angle greater than 60°.

In some embodiments, the means for agitating the tube is adapted toagitate the tube at an angle of from about 5° to about 60°, about 5° toabout 55°, about 5° to about 50°, about 5° to about 45°, about 5° toabout 40°, about 5° to about 35°, about 5° to about 30°, about 5° toabout 25°, about 5° to about 20°, about 5°, about 10°, about 15°, about20°, about 25°, about 30°, about 35°, about 40°, about 45°, about 50°,about 55°, or about 60°. As used herein, “agitation angle” and the likeare intended to refer to the angle measured from horizontal.

In some embodiments, the methods and systems described herein provide anavailable platelet count (“APC”) of greater than about 375,000platelets/microliter, about 400,000 platelets/microliter, about 425,000platelets/microliter, about 450,000 platelets/microliter, about 475,000platelets/microliter, about 500,000 platelets/microliter, about 525,000platelets/microliter, about 550,000 platelets/microliter, about 575,000platelets/microliter, about 600,000 platelets/microliter, about 625,000platelets/microliter, about 650,000 platelets/microliter, about 675,000platelets/microliter, about 700,000 platelets/microliter, about 725,000platelets/microliter, about 750,000 platelets/microliter, about 775,000platelets/microliter, about 800,000 platelets/microliter, about 825,000platelets/microliter, about 850,000 platelets/microliter, about 875,000platelets/microliter, about 900,000 platelets/microliter, about 925,000platelets/microliter, about 950,000 platelets/microliter, or about975,000 platelets/microliter.

In certain situations, the platelet count can not be calculated or isunreliable. These situations may be caused, for example, by anundesirable level of red blood cell infiltrate in the plasma portion ofthe separated sample. As used herein, the term “n/a” may refer tosituations in which the platelet count cannot be calculated or isunreliable.

Other embodiments provide methods for: suspending platelets in apost-centrifuged biological sample; increasing APC in a biologicalsample; and/or enriching the platelet count in a biological sample,comprising: centrifuging a collection tube containing a biologicalsample and a thixotropic gel; and agitating the collection tube at anangle and rate effective to create a layer of foam on top of saidbiological sample.

For avoidance of doubt, at least a portion of any one of the methodsdescribed herein could be suitable for use in any one of the systemsdescribed herein.

In some embodiments, the methods of the present invention furthercomprise the step of transferring a PRP sample to a microcentrifugationtube and spinning the tube (e.g. at about 2000 g) for a sufficient time(e.g. about 10 minutes) to remove cellular debris. Other embodiments ofthe present invention provide methods comprising the step of recoveringand freezing (e.g. at about −80 degrees C.) the supernatant. Stillfurther embodiments of the present invention provide methods comprisingthe step of analyzing the sample for growth factors using anenzyme-linked immunosorbent assay (ELBA).

In some embodiments, the methods of present invention provide anunexpected increase in growth factor levels when compared toconventional methods utilizing industry-standard instructions for use.Without being bound by theory, it is believed that the added physicalstimulus provided by certain embodiments of the present invention leadsto additional growth factor release from alpha granules, which could besupportive of better clinical outcomes.

Referring first to FIG. 1, an exemplary tube (100) containing abiological sample post-centrifugation is depicted. As shown therein, theplasma fraction (110) comprises platelet poor plasma (120) and plateletrich plasma (130), wherein the platelet rich plasma (130) has a portionof ultra-high platelet concentration, sometimes referred to asultra-high platelet rich plasma (140). Also depicted in FIG. 1 is theplasma-gel interface (150).

FIGS. 2 to 6 depict three exemplary agitation angles of the presentinvention and two comparative agitation angles. As discussed herein, theagitation angle is measured from the horizontal plane. In someembodiments, such as the one depicted in FIG. 4, the tube is agitatedalong a longitudinal axis (X).

Referring next to FIGS. 7 and 8, a tube (200) containing a biologicalsample after centrifugation and agitation in accordance with certainembodiments of the present invention is depicted. FIGS. 7 and 8 alsodepict the layer of foam (220) that appears on the top surface of theplasma fraction (210).

In contrast to FIGS. 7 and 8, tube (200) of FIG. 9 does not include alayer of foam on top of the plasma fraction (230).

Further areas of applicability of the present invention will becomeapparent from the detailed description provided hereinafter. It shouldbe understood that the detailed description and specific examples, whiledescribing exemplary embodiments, are intended for purposes ofillustration only and are not intended to limit the scope of the presentinvention.

EXAMPLES Example 1

A series of experiments were conducted to compare exemplary methods ofthe present invention to current methods of preparing PRP; and tounderstand how certain features impact platelet suspension and capture.In particular, agitation angle, agitation time, color of the biologicalsample and the presence of foam were evaluated. Change in color and thepresence of foam were evaluated at various time points during theexperiments. As described in Table 1 (below), experiments conducted withexemplary agitation angles and agitation times of the present inventionprovided surprisingly increased platelet counts with minimal to noinfiltration of unwanted cells from the biological sample (e.g.erythrocytes). The results of these experiments are described in Table 1(below).

TABLE 1 Agitation Platelet Agitation Time Count Method Angle (°)(seconds) Color (100K/μl) Whole Blood n/a n/a Deep red 193 Comp. Ex. 1*n/a n/a Straw 355 Comp. Ex. 2  15 60 Deep red n/a Comp Ex. 3  45 10Straw 397 Ex. 1 15 10 Straw w/pink hue 505 Ex. 2 15 30 Straw w/rose hue436 Ex. 3 45  5 Straw w/pink hue 416 Ex. 4 45 30 Straw w/rose hue 415Ex. 5 45 60 Light rose 480 Comp. Ex. 1 was a method performed inaccordance with techniques known in the industry, wherein the tube wasgently inverted horizontally twenty times.

As illustrated by the data described in Table 1 (above) the exemplarymethods of the present invention surprisingly increased platelet countswith acceptable levels of infiltrate. A foam layer was also observedwith each of the exemplary methods of the present invention. Withoutbeing bound by theory, the present inventors believe that agitationangle, agitation time and agitation rate are critical to achievingclinically maximal platelet counts. In addition, the appearance of foamon top of the plasma fraction provides a signal to the clinician thatthe desired platelet concentration has been achieved, as it correlateswith the increased platelet counts.

Example 2

Additional experiments were conducted to further demonstrate theincreased platelet counts provided by exemplary methods of the presentinvention. Five (5) samples from different donors were studied toevaluate the effect of the inventive methods at discrete time intervalsranging from five (5) seconds to one (1) minute. The impact of variousagitation angles, ranging from −15° to 90° from horizontal, were alsoevaluated. The impact of both fixed angle and swing-bucket centrifugeswas also evaluated. Platelet counts were performed using an automatedHoriba ABX Micros 60 Hematology Analyzer (Horiba Instruments, Inc.,Irvine Calif.).

The results of these experiments are described below in Tables 2 to 6.In each experiment 3 mL of platelet poor plasma (PPP) was removed beforethe platelets were counted in the PRP sample.

WB=Whole Blood

IFU=Instructions for Use

-   -   IFU platelet counts refer to platelet counts obtained using        techniques known in the industry, wherein the tube was gently        inverted horizontally twenty times.

TABLE 2 WB Platelet Count = 202/IFU Platelet Count = 241 SettingAgitation Time (Seconds) Centrifuge (speed × time) Angle 5 10 15 20 3040 50 60 Fixed 3300 × 10 −15 270 391 432 478 469 n/a Fixed 3300 × 10 0281 402 460 572 564 596 577 Fixed 3300 × 10 15 300 376 442 520 563 626607 Fixed 3300 × 10 45 266 358 302 371 n/a Fixed 3300 × 10 90 305 398n/a

TABLE 3 WB Platelet Count = 335/IFU Platelet Count = 437 SettingAgitation Time (Seconds) Centrifuge (speed × time) Angle 5 10 15 20 3040 50 60 Fixed 3300 × 10 −15 442 461 438 425 n/a Fixed 3300 × 10 0 498529 571 602 639 n/a Fixed 3300 × 10 15 501 535 581 672 602 628 638 649Fixed 3300 × 10 45 471 495 502 462 n/a Fixed 3300 × 10 90 521 n/a

TABLE 4 WB Platelet Count = 263/IFU Platelet Count = 358 SettingAgitation Time (Seconds) Centrifuge (speed × time) Angle 5 10 15 20 3040 50 60 Fixed 3300 × 10 −15 379 401 n/a Fixed 3300 × 10 0 421 482 508637 605 n/a Fixed 3300 × 10 15 439 521 595 639 678 654 638 Fixed 3300 ×10 45 444 507 582 604 n/a Fixed 3300 × 10 90 402 n/a

TABLE 5 WB Platelet Count = 321/IFU Platelet Count = 447 SettingAgitation Time (Seconds) Centrifuge (speed × time) Angle 5 10 15 20 3040 50 60 Swing 3300 × 10 −15 487 521 591 667 582 Swing 3300 × 10 0 598671 778 864 701 728 Swing 3300 × 10 15 608 788 901 853 846 Swing 3300 ×10 45 570 605 739 704 698 Swing 3300 × 10 90 683 721 629

TABLE 6 WB Platelet Count = 172/IFU Platelet Count = 304 SettingAgitation Time (Seconds) Centrifuge (speed × time) Angle 5 10 15 20 3040 50 60 Swing 3300 × 10 −15 387 419 601 662 583 539 Swing 3300 × 10 0408 488 573 701 745 705 728 Swing 3300 × 10 15 584 707 853 690 707 Swing3300 × 10 45 551 674 779 871 720 Swing 3300 × 10 90 402 336

As illustrated by the data described in Tables 2 to 6 (above), exemplarymethods of the present invention produce unexpected increases inplatelet counts when compared to the platelet counts produced byconventional methods. These differences are not only numericallysignificant, but they also provide a clinically significant advance tothe state of the art. Although the optimal time and angle may vary, thedata unequivocally show that the agitation method, across the range oftimes and angles studied, increased platelet counts, thereby increasingthe therapeutic dose of platelets that can be delivered to a subject.

Although several embodiments of the invention have been disclosed in theforegoing specification, it is understood by those skilled in the artthat many modifications and other embodiments of the invention will cometo mind to which the invention pertains, having the benefit of theteaching presented in the foregoing description and associated drawings.It is thus understood that the invention is not limited to the specificembodiments disclosed hereinabove, and that many modifications and otherembodiments are intended to be included within the scope of the appendedclaims. Moreover, although specific terms are employed herein, as wellas in the claims which follow, they are used only in a generic anddescriptive sense, and not for the purposes of limiting the describedinvention, nor the claims which follow.

What is claimed is:
 1. A method for separating components of a biological sample, the method comprising: introducing a biological sample having a plurality of components to a tube comprising: a lumen; a proximal end; a distal end; an interior wall; and an exterior wall; applying a centrifugal force to said tube for a time sufficient to separate said plurality of components; and agitating said tube at an angle of from 5° to 60°.
 2. (canceled)
 3. The method according to claim 1, wherein the tube further comprises a separation barrier.
 4. The method according to claim 3, wherein the separation barrier comprises a gel, a solid float or a combination thereof.
 5. The method according to claim 4, wherein the separation barrier comprises a gel and wherein the gel comprises a thixotropic gel. 6.-19. (canceled)
 20. The method according to claim 3, wherein the tube is agitated for a time sufficient to provide a plasma fraction having a straw color with a pinkish hue.
 21. The method according to claim 3, wherein the tube is agitated for a time sufficient to provide a plasma fraction having a hue angle, h, in the CIELAB system of from 310 to 350 degrees.
 22. The method according to claim 3, wherein the tube is agitated for a time sufficient to create a visually perceivable foam layer on a surface of the plasma fraction.
 23. (canceled)
 24. The method according to claim 22, wherein the foam layer has a thickness of from one millimeter to five millimeters.
 25. The method according to claim 24, wherein the foam layer has a density of from 0.01 g/cm³ to 0.25 g/cm³.
 26. The method according to claim 24, wherein the tube is agitated for from one (1) second to sixty (60) seconds.
 27. The method according to claim 26, wherein the tube is agitated at an angle of from 5° to 60°.
 28. The method according to claim 1, wherein the agitation is stepwise.
 29. The method according to claim 28, wherein the stepwise agitation comprises a plurality of five second intervals of agitation.
 30. The method according to claim 29, wherein the stepwise agitation further comprises a break between five second intervals.
 31. The method according to claim 30, wherein the break is from 0.1 seconds to 5 seconds. 32.-34. (canceled)
 35. The method according to claim 4, wherein the separation barrier comprises the gel, and wherein the centrifugal force creates a plasma-gel interface between a surface of the gel and a surface of the plasma fraction wherein the plasma-gel interface comprises platelets.
 36. (canceled)
 37. The method according to claim 35, wherein the agitation releases platelets from the plasma-gel interface.
 38. (canceled)
 39. The method according to claim 1, wherein the tube further comprises an anticoagulant. 40.-43. (canceled)
 44. A system for separating components of a biological sample comprising: a biological sample; a tube; a means for applying a centrifugal force to said tube; and a means for agitating said tube.
 45. The system according to claim 44, wherein the tube further comprises a separation barrier, optionally wherein the separation barrier comprises a gel, a solid float or a combination thereof.
 46. The system according to claim 45, wherein the gel comprises a thixotropic gel. 47.-57. (canceled)
 58. The system according to claim 44, wherein the system increases the APC by at least 10% versus the platelet count provided by a control system.
 59. (canceled)
 60. The system according to claim 58, wherein the means for agitating the tube is adapted to agitate the tube at an angle of from 5° to 60°.
 61. (canceled)
 62. The system according to claim 58, wherein the system provides a platelet count of at least 300,000 platelets/microliter. 63.-66. (canceled)
 67. A method for: suspending platelets in a post-centrifuged biological sample; increasing the number of platelets available for administration to a patient; and/or enriching the platelet count in a biological sample, comprising: centrifuging a collection tube containing a biological sample and a separation barrier; and agitating the collection tube at an angle and rate effective to create a thin layer of foam on top of said biological sample.
 68. The method according to claim 67, wherein the collection tube further comprises any one of the anticoagulants described herein.
 69. The method according to claim 67, wherein the biological sample comprises a plurality of components.
 70. The method according to claim 69, wherein the centrifugation is performed at a force of from 500 g up to 4000 g for a time sufficient to separate the plurality of components in the biological sample.
 71. The method according to claim 70, wherein the separation barrier comprises a thixotropic gel and wherein the thixotropic gel forms a barrier between the plurality of components of the biological sample.
 72. The method according to claim 67, wherein the biological sample comprises whole blood.
 73. The method according to claim 67, wherein the biological sample comprises a first component comprising a plasma fraction; and a second component comprising lymphocytes, monocytes and erythrocytes. 